去端肽胶原介导小干扰RNA抑制大鼠静脉移植物内膜增生

目的 评价局部应用组织因子小干扰RNA(siRNA)抑制静脉移植物内膜增生的效果.方法 SD大鼠120只,体重260～300 g,建立颈外静脉一颈总动脉移植模型后随机分成4组,每组30只.A组:去端肽胶原-TF StealthTMSelect RNAi组.B组:去端肽胶原-TF SteaLtHTM RNAi阴性对照组,C组:去端肽胶原组,D组:空白对照组.使用去端肽胶原-siRNA混合物涂抹于静脉移植物周围,免疫印迹检测术后1、3、7、14、28 d管壁组织因子蛋白表达,免疫组织化学法检测术后3 d管壁组织因子表达,同时检测术后14 d管壁增殖细胞核抗原指数,计算术后28 d新生内膜厚度.去端肽胶原-BLOCK-iTTM荧光寡核苷酸平行实验组12只,分别于术后3、7 d检测管壁BLOCK-iTTM寡核苷酸荧光确认其稳定性和转染效率.结果 平行实验组静脉移植物可观察到BLOCK-iTTM绿色荧光并持续到术后7 d.术后3 d A组siRNA有效抑制管壁TF蛋白表达,术后14 d A组与其他组比较增殖细胞核抗原指数明显减少(P＜0.05),术后28 d A组新生内膜厚度明显低于其他组(P＜0.05).结论 去端肽胶原携带siRNA可有效下调静脉移植物组织因子表达,抑制内膜增生.

Atelocollagen-mediated small interfering RNA delivery for effective gene silencing in rat vein grafts

Objective To evaluate the efficacy of using small interfering RNA targeting TF as a therapy for vein graft failure. Methods External jugular vein to carotid artery interposition vein grafts, which were applied to a low flow condition, were made in 120 Sprague-Dawley rats weighing 260 to 300 g. These rats were randomly divided into 4 groups, 30 rats each group. Group A was atelocollagen-TF Stealth<'TM> Select RNAi group. Group B was atelocollagen-TF Stealth<'TM> RNAi group. Group C was atelocollagen group. Group D was control group. Small interfering RNA mixed with atelocollagen was administrated to the external wall of grafted veins. The TF protein expression of vein gratis was analyzed by Western blot at 1, 3, 7, 14, and 28 d postoperatively, and by immunochemistry at 3 d postoperatively. The proliferation index was determined at 14 d postoperatively. Neointimal hyperplasia was evaluated at 28 d postoperatively. BLOCK-iTTM fluorescent oligo was used to confirm its stability and successful transfer into the vein graft wall at 3 and 7 d postoperatively for another group (n = 12). Results Fluorescence of BLOCK-iT<'TM> fluorescent align could be detected in the graft wall even at 7 d postoperatively. Knockdown of the TF expression was achieved by perivascular application of siRNA using atelocollagen. Compared with control group, the intima thickness at 28 d after grafting was significantly reduced (P ＜ 0.05). This phenomenon was preceded by significant reduction of cell proliferation in siRNA-treated grafts at 14 d postoperatively (P ＜0.05). Conclusion The expression of TF in vein grafts can be effectively inhibited by specific siRNAs using a atelocoilagen-based nonviral delivery approach/n vivo, so that the neointimal thickening can be prevented.